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Author ORCID Identifier

https://orcid.org/0000-0001-2345-6789

Date Available

5-25-2028

Year of Publication

2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Physiology

Faculty

Gregory Frolenkov

Faculty

Catalina Vélez-Ortega

Abstract

Sound perception relies on specialized actin-based structures called stereocilia, located on the apical surface of auditory hair cells (HCs). Stereocilia are arranged in three rows of increasing height and interconnected with extracellular tip links. Sound-induced vibrations modulate tension on these tip-links, leading to the opening or closing of mechanically gated ion channels that allow cation influx inside the cell, which ultimately generates a neural signal. In recent years, it has been established that the mechano-electrical transduction (MET) channel is constituted by an elaborate complex of multiple proteins associated with the channel itself and with the lower end of the tip-link. However, the molecular and biophysical mechanisms by which external forces are transmitted from the tip-link to the channel are yet unknown, along with the exact role of each protein of MET complex and the plasma membrane in this process.

This study represents the first systematic investigation of the MET channel activation in wild type HCs and HCs with deafness-associated variant of Calcium and Integrin-Binding protein 2 (CIB2). CIB2 is essential for hearing and MET in mammalian auditory HCs. CIB2 interacts with TMC1/2, the pore-forming subunits of the MET channel, and is needed for their localization to stereocilia. However, the molecular mechanism by which CIB2 regulates channel activity remains unclear. We generated a mouse model carrying the Cib2 p.R186W mutation that results in deafness but partially preserves MET currents in young postnatal auditory HCs. We found that the p.R186W variant increases resting open probability (Popen) of MET channels and steepens current-displacement relationships, while abolishing fast adaptation and slowing MET channel activation. This phenotype was not observed in Tmc1Δ/Δ OHCs, indicating that it is not due to altered pore identity. Instead, the slowed MET current kinetics and elevated Popen were consistent with loss of phosphatidylinositol 4,5-bisphosphate (PIP2) from stereocilia. The p.R186W mutation also disrupted localization of the membrane-associated protein BAIAP2L2, and electron-dense structures presumably linked to MET complexes. Pharmacological depletion of PIP2 and BAIAP2L2 in wild-type OHCs reproduced the p.R186W MET current phenotype but did not further affect activation and adaptation in Cib2R186W/R186W OHCs. We conclude that CIB2 is required for proper force transmission to MET channels by coordinating BAIAP2L2 and PIP2 localization at the mechanotransduction site.

This study addresses three key questions in the auditory field. First, developing an ultra-fast microfabricated actuator for stereocilia bundle deflection allowed, for the first time, to study systematically the molecular mechanisms underlying MET channel activation in the mammalian auditory hair cells. Second, we found that deafness-associated BAIAP2L2 modulates force sensitivity of the MET channels, most likely through its interactions with the plasma membrane. Finally, this work elucidates the molecular consequences of CIB2 mutations underlying Usher syndrome type 1J and non-syndromic deadness DFNB48, which may contribute to the development of future therapeutic treatments of these devasting diseases.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2026.309

Archival?

Archival

Funding Information

American Otological Society, Research Fellowship, 2024-2025

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